Recent investigations on cancer metabolism have inspired a number of new ideas for anti-cancer approaches. One such promising approach is to exploit the mechanisms governing the production of reactive oxygen species (ROS) in cancer. Some oncogenic mutations as well as tumor hypoxia are known to increase ROS production in cancer cells, which may play important roles in promoting tumor growth and metastasis. However, ROS is a double-edged sword, because too much ROS can also kill cancer cells. Cancer cells inside a solid tumor often reside in a hypoxic environment, meaning that the oxygen level is much lower than it is in normal tissues. Nonetheless, most anti-cancer agents are discovered using cancer cells maintained under a normal physiological concentration of oxygen. In hypoxic conditions, cancer cells exhibit oxidative phosphorylation (OXPHOS) defects and undergo metabolic reprogramming. Consequently, they become more aggressive and resistant to commonly used therapies. Hence, therapeutic approaches that can effectively eliminate hypoxic tumors are critically needed. Using a chemical screen, we have identified a compound that induces potent cytotoxicity toward a variety of cancer cells under hypoxic conditions and cancer cells carrying OXPHOS mutations. Intriguingly, our results further suggest a surge of mitochondrial ROS as the cause of cytotoxicity and point to a previously unrecognized ROS-producing enzyme complex in OXPHOS- defective cancer cells. Moreover, we have shown that, in combination with other agents currently being used in human, our candidate compound can become effective against a broad spectrum of cancer types even in normoxic conditions. The selected agent has been shown to confer cadioprotection in mice and extend lifespan in worms, suggesting good safety profiles in vivo. Thus, the major goals of this application are to unravel the controls of this newly discovered ROS-producing machinery in cancer, to investigate the therapeutic potential of the selected agent in mice and to identify the cancer types most susceptible to these new approaches. By successfully completing the proposed research, we hope to gain critical knowledge that may lead to nultiple effective and broadly applicable anti-cancer treatment options.